Bacterial DNA initiates inflammatory responses and is responsible for development of some level of innate immunity in mammals (Ashkar et al., 2002, Curr. Mol. Med. 2: 545-556; Pisetsky, 1999, Immunol. Res. 19: 35-46; Krieg et al., 1995, Nature 374: 546-549; Krieg, 1999, J. Gene Med. 1: 56-63). The oligodeoxynucleotide (ODN) ligand specificities of mammalian NK cells, antigen presenting cells (B-cells, macrophages, dendritic cells) and T-cells have been extensively reviewed (Krieg, 2000, Curr. Opin. Immunol. 12:35-43; Krieg, 1999, Biochim. Biophys. Acta 1489:107-116; Pisetsky et al., 1999, Biochem. Pharmacol. 58:1981-1988; Pisetsky, 1996, Immunity 5:303-310; Pisetsky et al., 1993, Mol. Biol. Rep. 18:217-221; Krieg, 2000, Vaccine 9:618-222; Scheule, 2000, Adv. Drug Deliv. Rev. 44:119-134, Weiner, 2000, J. Leukoc. Biol. 68:455-463, Lipford et al., 1998, Trends Microbiol. 6:496-500).
The chemistry and conformation of single base phosphodiester (Po) oligodeoxyguanosine was unique compared to other single base oligos. Po (dG30 (only) (SEQ ID NO:1) and Ps single base ODNs were mitogenic for B-cells (Pisetsky et al., 1993, Mol Biol Rep. 18:217-221). Interestingly, negative immunoregulatory activities were also reported for single base ODNs as well as for CpG (SEQ ID NO:7) ODNs. Pretreatment of J774 cells with Po dG30 (SEQ ID NO:1) inhibited E. coli DNA, LPS and CpG (SEQ ID NO:7) activation of IL-12 (Pisetsky et al., 1993, Mol. Biol. Rep. 18:217-221) and nitric oxide (Zhu et al., 2002, J. Leukoc. Biol. 71:686-694) production. All other Po single base ODNs (i.e. dA, dT, dC) tested did not produce this effect. It appears that single base ODNs may have different effects based on whether they are Ps or Po and on the target cell type involved in the immune response (Zhu et al., 2002, J. Leukoc. Biol. 72:1154-1163).
Other studies of the effects of unique nucleic acids on immune function have been published. Conjugation of dG “Runs” to CpG (SEQ ID NO:7) (i.e. dG6 (SEQ ID NO:6) produced an ODN that bound mouse APC (Lee et al., 2000, J Immunol 165:3631-3639), and CD8 positive T-cells (Lipford et al., 2000, Immunology 101:46-52). In these studies, binding was followed by activation as shown by secretion of TNF-alpha and IL-12 (Lipford et al., 2000, Immunology 101:46-52) as well as initiation of T-cell proliferation and cytotoxicity (Lee et al., 2000, J Immunol 165:3631-3639). Conjugation of dG “Runs” to 3′ and 5′ ends of CpG ODN (SEQ ID NO:7) enhanced NK cell lytic activity (Ballas et al., 1996, J. Immunol. 157:1840-1845). Costimulatory effects of guanosine-rich (G-rich) ODNs were shown in T-cells where costimulation by G-rich ODNs induced CTL activity (Lipford et al., 1998, Trends Microbiol. 6:496-500). CpG (SEQ ID NO:7) palindromes containing flanking sequences or “runs” of 12 guanosine nucleotides (i.e. dG12 (SEQ ID NO:2) bind to and activate mouse NK cells (Kimura et al., 1994, J. Biochem. 116:991-994).
There are several different classes/types of DNA binding proteins on mammalian cells represented by a limited number of germ-line encoded receptors that are expressed predominantly on antigen presenting cells (with low levels of expression on T-cells and NK cells). These proteins are referred to as pattern recognition receptors (PRR) (Krug et al., 2003, J. Immunol. 170: 3468-3477; Sano et al., 2003, J. Immunol. 170: 2367-2373) and they bind to pathogen associated molecular pattern ligands (PAMP) of microbial origin. The PAMPS include LPS, peptidoglycan, certain lipoproteins, CpG oligodeoxynucleotides (ODNs) (SEQ ID NO:7), etc. The most widely distributed of the PRR that bind ODNs are the Toll-like receptor 9, Mac-1 (CD 11b/CD18) (Stacey et al., 2000, Curr. Top. Microbiol. Immunol. 247: 41-58; Takeda et al., 2003, Ann. Rev. Immunol. 21: 335-376; Hemmi et al., 2000, Nature, 408: 740-745, Benimetskaya, 1997, Nat. Med. 3: 414420; Bauer et al., 2001, J. Immunol. 166: 5000-5007) and Scavenger receptor-A (Kimura et al., 1994, J. Biochem. (Tokyo) 116:991-994; Peiser et al., 2002, Curr. Opin. Immunol. 14:123-128). ODN binding to PRR may cause either activation or inhibitory responses depending on the ODN concentration and target cell type. In addition, ligation of PRR in vivo by ODNs may also produce different pathways of immunoregulation such as autoimmunity, Th1 bias activation, etc. (Bauer et al., 2001, Proc. Natl. Acad. Sci. U S A. 98: 9237-9242; Chuang et al., 2002, J. Leuk. Biol. 71: 538-544; Kerkmann et al., 2003, J. Immunol. 170: 4465-4474).
Antimicrobial Proteins and Peptides
Naturally occurring antimicrobial proteins and peptides (AMP) have been identified from plant, invertebrate and vertebrate species (Hancock et al., 2000, Trend Microbiol., 8:402-410; Hancock et al., 1995, 37:135-275; Hanson et al., 2000, In: Cytotoxic Cells: Basic Mechanisms and Medical Application, M V Sitkovsky and P A Henkart (eds), Lippincott Williams and Wilkins, Phil, Pa. 213-227; Vizioli et al., 2000, Trends Pharmacol. Sci., 23: 494-496; Zhai et al., 2000, Biochim. Biophys. Acta 1469: 87-99). These AMPs have been classified based on both chemical and conformational properties and they can be differentiated based on whether the active form is a peptide (i.e. 17-35 aa in length) or a protein (>50 aa). An additional distinguishing property of AMP is their cationic nature with little to no amino acid sequence identity across all the members of this very large group. For example, cecropins, magainins and defensins from silk moth, Xenopus and mammals (respectively) are all low mw AMP. Although they share lysine rich regions and are inducible, they have no sequence homology. The functional characteristic of this large group of AMP is based on their common ability to kill bacteria and (in some cases) eukaryotic cells.
The amino acid content of these AMPs provides clues regarding the common chemical and physical features that may be responsible for their bactericidal effects. An example is the recently described AMP Cupiennin-1 (Kulm-Nentwig et al., 2002, J. Biol. Chem., 13: 11208-11216). This 35 aa basic peptide has 8 lysine residues, is present in the venom of Cupiennius salei (a hunting spider found in Central America), is amphipathic and has bactericidal activities against Gram negative and Gram positive bacteria. This peptide may be similar to other AMPs (e.g. magainins; Jacob et al., 1994, Ciba Foundation Symposium 186:197-216) regarding the mechanism of binding to bacterial cells. It was predicted to fold into an amphipathic alpha-helix when it inserts into the bacterial cell membrane. Differential sensitivities of eukaryotic versus prokaryotic cells are thought to be based on the low cholesterol content and relatively high negative charge density of bacterial cell walls compared to eukaryotic cells (Jacob et al., 1994, Ciba Foundation Symposium 186: 197-216).
Another type of AMP is not naturally occurring, but is generated in vitro by proteolytic digestion or acid hydrolysis of some precursor, larger mw molecule. These AMP are relevant innate immune response effector substances. One interesting class that has been studied is histone like proteins. The traditional cellular location of histone proteins (H1) is in the nucleus associated with chromatin fibers either in the form of linker histone 1 or core histones (H2a, H2b, H3 and H4) that form nucleosomes. However, studies performed in higher vertebrates have shown that many cells of the immune system express cytoplasmic and membrane forms of these proteins (Ojcius et al., 1991, Immunol. Lett., 28: 101-108; Watson et al., 1994, Biochem. Soc. Trans. 22:199S; Watson et al., 1995, Biochem. Pharmacol., 50: 299-309; Holers et al., 1985, J. Clin. Invest. 76: 991-998; Bennett et al., 1985, J. Clin. Invest. 76: 2182-2190; Emlen et al., 1992, J. Immunol. 148: 3042-3048; Watson et al., 1995, Biochem. Pharm. 50: 299-309; Bolton et al., 1997, J. Neurocytology 26: 823-831; Bennet et al., 1988, J. Immunol. 140: 2937-2942; Rose et al., 1998, Inf. Immun. 66: 3255-3263; Eggena et al., 2000. J. Autoimm. 14: 83-97; Kubota et al., 1990, Immunol. Lett. 23:187-193). The function(s) of histone like membrane proteins (HLMP) cationic proteins have generally not been ascribed to ligand or receptor activities except for thyroglobulin binding by an H1 receptor on mouse macrophages (Brix et al., 1998, Clin. Invest. 102: 283-293) and DNA binding by a 28 kDa protein on “normal” human lymphocytes (Gasparro et al., 1990, Photochem. Photobiol., 52: 315-321). Evidence for the association of cell-derived. and/or cell membrane histone H1 as a participant in antibacterial innate immunity has also been provided by studies of human ileal mucosal extracts (Rose et al., 1998, Inf. Immun. 66: 3255-3263) and human ulcerative colitis (UC) (Eggena et al., 2000. J. Autoimm. 14: 83-97). In both cases H1 was either released from villus epithelial cells (Rose et al., 1998, Inf. Immun. 66: 3255-3263) or was associated with a serum marker for UC (Eggena et al., 2000, J. Autoimm. 14: 83-97). In addition, Raji cells express 14, 17, 18, 33 and 34 kDa DNA binding proteins (Kubota et al., 1990, Immunol. Lett. 23: 187-193). These histone or histone-like proteins were described as being responsible for the binding, endocytosis and degradation of exogenous DNA. Interestingly, the thyroglobulin receptor on the cell surface of J774 (mouse) macrophages is an H1 protein (Brix et al., 1998, J. Clin. Invest. 102: 283-293).
Teleost Derived Immune Activity
Histone proteins and peptides with antimicrobial activities have been isolated from various teleosts, e.g., salmon blood, liver, intestine and mucus (Patrzykat et al., 2001, Antimicrob. Agents Chemother, 45:1337-1342). Catfish skin, epithelial cells and mucus contain H2A-like (Parasin-I) and H2B-like molecules (Cho et al., 2002, FASEB J. 16: 429-431). Histone release from teleost cells require tissue injury; thus, membrane expression of histone-like proteins was not determined. CpG (SEQ ID NO:7)-induced activation of rainbow trout macrophages was determined by induction of IL-1β and IFN-like cytokines (Jorgensen et al., 2001, Fish Shellfish Immunol., 8: 673-682). Similarly, CpG (SEQ ID NO:7) activated leukocytes from Atlantic salmon had increased interferon production (Jorgensen et al., 2001, Dev. Comp. Immunol., 4: 313-321).
Nonspecific cytotoxic cells from catfish have been found to be activitated by bacterial DNA and ODN's (Oumouna et al., 2002, Dev. Comp. Immunol. 26:257-269). Cellular activation resulted from the binding of synthetic ODNs (sODN) to NK-like nonspecific cytotoxic cells (NCC). Differences were described in the teleost cells compared to mammalian “canonical” dogma. The preferred binding motifs for mammalian cells consist of -GACGTT- (mice) and -GTCGTT- (humans) (Krieg A M., 2002, Curr. Opin. Immunol. 12:35-43) and GpC dinucleotides do not bind these cells. The optimum immunostimulatory motif for teleosts was composed of either 5′-C/AT/AGCTT-3′ or 5′-GTCGTT-3′ (Oumouna et al., 2002, Dev Comp Immunol 26:257-269). Methylation of cytosine inhibited teleost activation responses to sODNs. NCCs were activated by sODNs containing dinucleotides flanked by consecutive deoxyguanosine residues (dG runs) in addition to the single base oligodeoxyguanosine 20-mer nucleotide (i.e. dG20 (SEQ ID NO:6)) (Oumouna et al., 2002, Dev. Comp. Immunol. 26:257-269).
Studies have been conducted to determine if SR-A-Type-I was responsible for dG20 (SEQ ID NO:6) binding activity to NCC (Kaur et al., 2003, Fish Shellfish Immunol. 15:169-181). Those results demonstrated that total binding by dG20 (SEQ ID NO:6) to NCC could not be explained solely by expression of Scavenger Receptor-A (SR-A) because antibody to SR-A or SR-A ligands (i.e. dextran sulfate, polyvinyl sulfate) could only compete 40-50% of total dG20 (SEQ ID NO:6) binding. However, antimicrobial proteins have been isolated from Atlantic salmon (Salmo salar) (Richards et al., 2001, Biochem. Biophys. Res. Comm. 284: 549-555; from striped bass (Noga et al., 2001, Parasitol. 123: 57-65; U.S. Pat. No. 6,753,407); Coho salmon and flounder (Patizkat et al., 2001, Antimicrob. Agents Chemother. 45: 1337-1342); rainbow trout (Fernandez et al., 2002. Biochem. J. 368: 611-620); from catfish (Park et al., 1998, FEBS Lett. 437: 258-262; U.S. Pat. No. 6,316,594; Cho et al., 2002, FASEB J. 16: 429-431); from tiger shrimp (Peaeus monodon) (U.S. Pat. Appl. Pub. No. 2004/0235738) for model studies of the role of histone-like proteins in antimicrobial immunity).
Toll-like receptor proteins have not yet been identified on teleost cells although a trout homologue has been obtained from an EST library of differentially expressed liver genes (Bayne et al., 2001, Dev. Comp. Immunol. 25: 205-17). However, its function remains unknown. Similarly, two additional EST's from zebrafish (Accession numbers: BF158452 and BG304206) have identified fragments of Toll-like genes, but their functions also remain unknown. Complete sequencing and functional characterization of these molecules and other PRR will provide invaluable insight into the evolution of these receptors and their role in pathogen resistance.